Synthetic rhodamine 110 derivative substrates heretofore have been utilized for fluorometric measurement of protease enzyme activity, as reported by Leytus et al., Biochem. Journal 209: 299-307 (1983) and Leytus et al., Biochem. Journal 215: 253-260 (1983). The Leytus et al., substrates were extremely sensitive for measurement of enzyme activities, as compared to previously reported substrates. However, the Leytus et al. rhodamine 110 derivative substrates have been found to exhibit low water solubility of about 120 micromolar at 37.degree. C., thereby necessitating the addition of organic solvent additives, e.g. dimethylformamide and ethanol, for promoting the substrate solubility. However, addition of organic solvents to enzyme assays is known to interfere with the assay procedure, i.e. denaturing the protein, decreasing reaction rate and/or causing precipitation of the other reactants or products, resulting in less than optimum test performance.
In addition to the disclosure of Leytus et al., a coumarin derivative substrate for the fluorometric and spectrophotometric determination of proteolytic enzymes in biological fluids was disclosed by Smith et al., U.S. Pat. No. 4,294,923. The Gargiulo et al. U.S. Pat. Nos. 4,275,153 and 4,336,186 disclose a fluorogenic aminoisophalate substrate for determination of protease enzyme activities in biological samples.